Continuously emerging new and hard-to-treat microbes, and the growing incidence of multi-drug resistant infections pose formidable challenges to human health. Innovative approaches are urgently needed to speed up the discovery of new anti-infectives. Our aim is to achieve a paradigm shift in antimicrobial drug discovery by finding next generation anti-infectives that prevent disease by blocking pathogen adaptation to host physiology. To this end we propose using whole live animals for high throughput screening of small molecules. We have developed infection models in the nematode Caenorhabditis elegans that can be used to identify drugs that cure otherwise lethal infections. High throughput screening of nematodes in 384-well plates is followed by secondary screening in a more highly evolved model host, the fruit fly Drosophila melanogaster, increasing the likelihood of isolating drugs that will work in humans. Our approach is applicable to many different classes of microorganisms, including bacteria, viruses, fungi and parasites. It has several advantages over traditional drug discovery: (i) In addition to identifying conventional antibiotics, it will uncover entirely new classes of anti-infectives that only exhibit in vivo activity. Examples are "virulence blockers" and "immune escape blockers". (ii) Our approach is unbiased and requires no prior knowledge of potential drug targets or pathways. (iii) It bypasses the current bottleneck of toxicity/efficacy testing by automatically eliminating toxic compounds (because they would kill the nematodes), yielding quality hits with in vivo activity. (iv) It will identify compounds that prevent or mitigate microbial resistance development, or can be combined with antibiotic therapy, thereby increasing antibiotic efficacy. We predict that our approach can identify compounds that inhibit diverse aspects of virulence: (i) adhesion and colonization, (ii) epithelial barrier disruption, (iii) deep tissue invasion, (iv) biofilm formation, (v) avoidance of immune recognition, and (vi) modulation of immune signaling. Some of the molecular mechanisms underlying these processes are conserved across bacterial species. To establish proof-of-principle, we seek funding for discovering new anti-infectives against Pseudomonas aeruginosa, one of several gram-negative bacteria that have recently emerged in a multi-drug resistant form for which efficient antibiotics are either limited or not available. We plan to screen a large number of chemical compounds (250,000) to maximize the discovery of new classes of anti-infectives. Promising compounds will undergo characterization, efficacy testing in other gram-negative bacteria (Klebsiella, Acinetobacter, Enterobacter) and testing in mouse models of infection. For highly promising candidates we will attempt molecular target identification. PUBLIC HEALTH RELEVANCE: Microbes that cause disease are becoming resistant to antibiotics faster than we can find new ones, making many common infections untreatable and life threatening. The goal of our project is to find a way to identify a new generation of antibiotics. Rather than simply preventing bacteria from growing, these new sophisticated drugs will prevent disease by interfering with a microbe's ability to interact with the human body.